Identification of cellular receptors for flaviviruses will help provide an understanding of the pathogenesis of diseases caused by these agents. Several approaches were taken to the identification of cellular receptors for DEN viruses: (1) Virus overlay protein binding assay (VOPBA). Proteins in lysates of cells (human skin cells, LLCMK2 cells, C6/36 cells, etc.) are electrophoretically separated, the resulting gel is blotted onto nitrocellulose (or nylon or other membrane). Radio-labeled virus particles are prepared and incubated with the blot in the expectation that virus particles will bind to their receptor molecule(s) on the blot. This strategy was successfully used to identify the receptor for mouse hepatitis virus, a coronavirus, but was not successful for dengue virus. (2) "SOPBA" (membrane proteins and virus particles are cross-linked in solution). Cell are radio-labeled and membrane proteins are prepared. Proteins are cross- linked using DTSSP. DEN E-specific antibodies are added to immune precipitate the viral receptor (E) bound to its cellular counterpart. RIP'd, E-related proteins are separated by SDS-PAGE to identify an E- specific band that was radiolabeled and shifted in size due to its cross- linking to a receptor molecule. This strategy was also abandoned. (3) Molecular approaches. We sought to identify a cell line that lacked a dengue virus receptor but was otherwise competent for production of infectious progeny virions. Such cells could be used to express foreign DNAs (derived from receptor-bearing cells) which might encode a receptor. Normal Rat Kidney cells were initially chosen for analysis, since dengue viruses do not replicate in them. NRK cells were transfected with infectious cDNA for DEN4 virus (obtained from Dr. C.J. Lai), thus bypassing the need for a receptor to initiate infection. Transfected NFK cells were mixed with permissive LLCMK2 cells in an "infectious center" assay. Results suggested that NRK cells are blocked for replication of dengue viruses at a stage following virus entry. Similar experiments with different cell types are in progress.